Compounds and methods for enhancing erythropoiesis

ABSTRACT

Compounds and methods for enhancing erythropoiesis. The compound contains a chemical structure of the formula (I) indicated below, in which R is a glucosyl group. In addition to having an erythropoiesis effect, the compound of the formula (1) is effective in enhancing erythropoietin formation, and increasing kidney function and expression of hepatocyte growth factor. The method includes the step of administering an effective amount of the compound of the formula (I) to a subject in need thereof and thereby results in an enhancement of erythropoiesis.

FIELD OF THE INVENTION

The present invention relates generally to erythropoiesis, and morespecifically to enhancement of erythropoiesis.

BACKGROUND OF THE INVENTION

Chronic kidney disease (CKD) is a worldwide public health problem withadverse outcomes of kidney failure, cardiovascular disease and prematuredeath (Levey, 2005). Patients with CKD are at high risks for progressionto the end stage renal disease and need the dialysis or kidneytransplantation to maintain a long-term survival. Anemia, an earlysymptom of CKD, results from underproduction of endogenouserythropoietin (Epo) by kidney (Zarzecki el al., 2004). In addition toCKD, anemia is also associated with other diseases, such as cancer,acute and chronic infections, autoimmune, inflammation and chronicrejection after solid-organ transplantation (Weiss and Goodnough, 2005).

Epo is a glycoprotein hormone mainly produced in adult kidney and fetalliver. Epo exerts its effect by binding to erythropoietin receptor (Eporeceptor) on cell surface. When cells sense a relatively low oxygenlevel (such as the hypoxia), Epo is produced and released to regulateproliferation, differentiation, maturation, and survival in erythroidlineage cells (Moritz e al., 1997 and Fisher, 2003). Abnormal Epo levelsin bloodstream may be an indicator for bone marrow and renal diseases.Relatively low Epo levels have been seen in patients with CKD, primarypolycythemia rubra vera and chemotherapy-induced anemia. Relatively highEpo levels have been seen in secondary polycythemia and renal cancerpatients (Eckardt and Kurtz, 2005 and Hodges el al., 2007).

Besides being produced in kidney and liver tissues, Epo and itsreceptors have been found in non-erythroid tissues and organs, includingbrain, eye, heart, lung, gut, pancreas, muscle, uterus and gonads(Eckardt and Kurtz, 1992). Epo-Epo receptor signaling contributes towound healing responses, angiogenesis and local tissue-protectivefunctions, such as neuroprotections, cardiovascular protections andprotections from tissue ischemia and ischemia/reperfusion injury(Pascilos el al., 2008 and Arcasoy, 2008). It has been reported that Epohas renoprotective effects by reducing the extent of renal dysfunctionand facilitating the recovery from cisplatin-induced acute renal failure(Sepodes et al., 2006 and Arcasoy, 2008).

Erythropoiesis-stimulating agents (ESAs) are recommended by the NationalKidney Foundation Kidney Disease Outcomes Quality Initiative guidelinesto treat anemia of CKD in patients with treatment-responsive anemia.Recombinant human Epo (rHuEpo) has been approved for treating anemia ofCKD, anemia in cancer patients receiving chemotherapy, for reducingtransfusion requirements during surgery and for treating anemia inzidovudine-treated patients infected with human immunodeficiency virus.A novel erythropoiesis-stimulating protein (NESP), designed from Epowith a longer plasma half-life, has been approved for treating anemia inchronic renal failure (Fisher, 2003). It has been recommended thatintravenous (i.v.) or subcutaneous (s.c.) administration of NESP be morethan once per week for maintenance therapy. The pain, inconvenience dueto frequent injections, and development of anti-Epo antibodies becauseof inherent antigenicity associated with rHuEpo are of a great concerned(Bunn, 2007). Moreover, ESAs pose safety risks in patients with higherhemoglobin levels and may cause complications such as hypertension,thromboembolism, iron deficiency and severe pure red-cell aplasia (Wishand Coyne, 2007).

Therefore, a heretofore unaddressed need exists in the art to addressthe aforementioned deficiencies and inadequacies, especially inconnection with erythropoiesis and kidney functions.

SUMMARY OF THE INVENTION

In one aspect, the invention is related to a method for enhancingerythropoietin formation in a subject in need thereof. The methodincludes administering to the subject an effective amount of a compoundof the formula:

wherein R is a glycosyl group.

The glycosyl group is at least one selected from the group consisting ofdihydroxyacetone, glucose, galactose, glyceraldehydes, threose, xylose,mannose, ribose, ribulose, xylulose, tagatose, psicose (allulose),fructose, sorbose, rhamnose, erythrose, erythrulose, arabinose, lyxose,allose, altrose, gulose, idose and talose and any combinations thereof.

In another aspect, the invention is related to a method for treating adisease and/or disorder in a subject that would benefit fromerythropoietin treatment. The method includes administering to thesubject an effective amount of the aforementioned compound of theformula (I).

The disease is at least one selected from anemia, a renal failure, anderythropoiesis deficiency-related diseases.

The disorder would benefit from an erythropoietin-related protectiveeffect.

The erythropoietin-related protective effect is at least one selectedfrom neuroprotection, cardiovascular protection, renoprotective effect,and protections from tissue ischemia and ischemia/reperfusion injury.

In another aspect, the invention is related to a method for enhancingerythropoiesis in a subject in need thereof. The method includesadministering to the subject an effective amount of the aforementionedcompound of the formula (I).

Further in another aspect, the invention is related to a method forenhancing kidney function in a subject in need thereof. The methodincludes administering to the subject an effective amount of theaforementioned compound of the formula (I).

Yet in another aspect, the invention is related to a method forincreasing the expression of hepatocyte growth factor in a subject inneed thereof. The method includes administering to the subject aneffective amount of the aforementioned compound of the formula (I).

In another aspect, the invention is related to a method for treating adisease and/or disorder in a subject that would benefit from hepatocytegrowth factor treatment. The method includes administering to thesubject an effective amount of the aforementioned compound of theformula (I).

Further in another aspect, the invention is related to a method fortreating a disease and/or disorder via regulating the expression ofhepatocyte growth factor in a subject in need thereof. The methodincludes administering to the subject an effective amount of theaforementioned compound of the formula (I). The disease and/or disorderis at least one selected from allergic inflammation, osteoarthritis,rheumatoid arthritis, muscular dystrophy, muscular atrophy, skin ulcer,burn, scleroderma,.crush syndrome, cerebrovascular diseases such astransient ischemic attack and stroke, neurodegenerative diseases such asamyotrophic lateral sclerosis, Alzheimer's disease and, Parkinson'sdisease, spinal cord injury, diabetic retinopathy, peripheralneuropathy, spinal canal stenosis, deafness, acute hepatitis, livercirrhosis, fulminant hepatitis, fatty liver, surgical treatments forliver transplantation, partial resection and ischemia, kidney fibrosis,acute renal failure, chronic renal failure such as nephritic syndromeand obstructive nephropathy, surgical treatments for renaltransplantation and ischemia, diabetic nephropathy, acute pneumonia,pulmonary fibrosis, surgical treatments for lung transplantation,partial resection and ischemia, inflammation-mediated heart diseasessuch as cardiac allograft rejection and myocarditis, cardiovasculardiseases such as angina, cardiac infarction, cardiomyopathy andatherosclerosis obliterans, intestinal mucosal injury, inflammatorybowel disease, gastric ulcer or diabetes mellitus.

These and other aspects will become apparent from the followingdescription of the preferred embodiment taken in conjunction with thefollowing drawings, although variations and modifications therein may beaffected without departing from the spirit and scope of the novelconcepts of the disclosure.

The accompanying drawings illustrate one or more embodiments of theinvention and, together with the written description, serve to explainthe principles of the invention.

Wherever possible, the same reference numbers are used throughout thedrawings to refer to the same or like elements of an embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing Compound A enhances hemoglobin levels in bonemarrow cells.

FIG. 2 is a graph showing Compound A stimulates erythroid progenitorcell proliferation using burst-forming units-erythroid (BFU-E) assay onbone marrow cells.

FIG. 3 is a graph showing Compound A increases the expression oferythropoietin (Epo) in kidney tissue.

FIG. 4 is a graph showing Compound A increases the expression oferythropoietin (Epo) in hepatocytes.

FIG. 5 is a graph showing Compound A increases the number of red bloodcells in a cisplatin-induced acute renal failure animal model.

FIG. 6 is a graph showing Compound A decreases serum blood urea nitrogenlevels in a cisplatin-induced acute renal failure animal model.

FIG. 7 is a graph showing Compound A increases the number of red bloodcells in a cisplatin-induced acute renal failure animal model at day 23.The horizontal arrow represents Compound A treatment (mg/Kg/day).

FIG. 8 is a graph showing Compound A decreases serum blood urea nitrogenlevels in a cisplatin-induced acute renal failure animal model at day23. The horizontal arrow represents Compound A treatment (mg/Kg/day).

FIG. 9 is a graph showing Compound A increases the expression oferythropoietin (Epo) in liver tissue in a cisplatin-induced acute renalfailure animal model. The horizontal arrow represents Compound Atreatment (mg/Kg/day).

FIG. 10 is a graph showing Compound A increases the expression ofhepatocyte growth factor (HGF) in liver tissue in a cisplatin-inducedacute renal failure animal model. The horizontal arrow representsCompound A treatment (mg/Kg/day).

FIG. 11 is a graph showing Compound A stimulates erythroid progenitorcell proliferation using burst-forming units-erythroid (BFU-E) assay onbone marrow cells in a cisplatin-induced acute renal failure animalmodel. The horizontal arrow represents Compound A treatment (mg/Kg/day).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. For convenience, certainterms may be highlighted, for example using italics and/or quotationmarks. The use of highlighting has no influence on the scope and meaningof a term; the scope and meaning of a term is the same, in the samecontext, whether or not it is highlighted. It will be appreciated thatsame thing can be said in more than one way. Consequently, alternativelanguage and synonyms may be used for any one or more of the termsdiscussed herein, nor is any special significance to be placed uponwhether or not a term is elaborated or discussed herein. Synonyms forcertain terms are provided. A recital of one or more synonyms does notexclude the use of other synonyms. The use of examples anywhere in thisspecification including examples of any terms discussed herein isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to various embodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control.

As used herein, “around”, “about” or “approximately” shall generallymean within 20 percent, preferably within 10 percent, and morepreferably within 5 percent of a given value or range. Numericalquantities given herein are approximate, meaning that the term “around”,“about” or “approximately” can be inferred if not expressly stated.

EXAMPLES

Without intent to limit the scope of the invention, exemplaryinstruments, apparatus, methods and their related results according tothe embodiments of the present invention are given below. Note thattitles or subtitles may be used in the examples for convenience of areader, which in no way should limit the scope of the invention.Moreover, certain theories are proposed and disclosed herein; however,in no way they, whether they are right or wrong, should limit the scopeof the invention so long as the invention is practiced according to theinvention without regard for any particular theory or scheme of action.

Example 1 Purification of Compound A

Two kilograms of dry Polygonum multiflorum were ground in a pulverizerand the ground material was immersed in 2 liters of 85% (v/v) ethanolovernight to form a reaction solution. The reaction solution wascollected, and another 2 liters of 85% (v/v) ethanol were added to theground Polygonum multiflorum residue for further extraction according tothe aforementioned procedure. The ethanol extraction was repeated foradditional three times. The collected solution was filtrated using agas-extracting apparatus (with Whatman #1 filter papers), and followedby concentration in a rotavapor (Buchi) at 40° C. to a six-foldreduction in volume. The concentrated filtrates were pooled together andpartitioned 5 times with n-hexane/H₂O=1:1 (total 5 liters). The aqueousphase was collected and further partitioned 6 times with ethyl acetatewater, EtOAc/H₂O=1:1 (total 6.5 liters), to obtain EtOAc layer. SixEtOAc layers were pooled together, concentrated, and the concentrate wasdried in Freeze Dryer (Kingniech, Taiwan) to obtain the EtOAc extract(called “PoMuEPe”). The PoMuEPe was chromatographed on a Diaion® HP-200column (4.8×60 cm; Mitsubishi Chemical) with a Methanol:H₂O=50:50solvent system at a flow rate of 2 ml/min. Fractions were collected,dried and assayed for the ability to enhance hemoglobin formation.Fractions exhibiting activities were collected and called PoMuEPeD9.PoMuEPeD9 was chromatographed on a Sephadex™ LH20 column (2.6×100 cm; GEHealthcare Life Science) with a Methanol:H₂O=100:0 solvent system at aflow rate of 1 ml/min. Fractions were collected and assayed for theirabilities in enhancing hemoglobin formation. Fractions showingbioactivity were collected and called PoMuEPeD9L11.

The chemical structure of the major component in PoMuEPeD9L11 wasdetermined by nuclear magnetic resonance (NMR) spectroscopy (Bruker) andliquid chromatography/Mass spectrometry (LC/MS; Bruker). It wasidentified that the major component was 2,3,5,4′-tetrahydroxystilbene2-O-β-D-glucopyranoside, called “Compound A,” having the formula:

wherein R is a glucosyl group.

Example 2 Compound A Enhances Hemoglobin Formation

C57BL/6JNar1 mice, 8-10 weeks of age, were purchased from the NationalLaboratory Animal Center (NLAC, Taiwan). Acute hemolytic anemia wasinduced by a single intraperitoneal (i.p.) injection of phenylhydrazinehydrochloride (Sigma-Aldrich) at a dose of 100 mg/kg in a phosphatebuffered saline (PBS). Six days after the injection, bone marrow cellswere isolated from mice and cultured according to the proceduredescribed in Worthington el al., (I 985) and Rosenthal et al. (1987)with minor modifications. The cell suspension was adjusted to a densityof about 6×10⁵ cells/ml in MEM alpha medium (α-MEM, Gibco) containing 1%(v/v) bovine serum albumin (BSA, Sigma-Aldrich), 7.5 μM of2-mercaptoethanol (Sigma-Aldrich), 1.4 mM of L-glutamine(Sigma-Aldrich), 10 μM of ferric chloride (FeCl₃, Sigma-Aldrich) and 50mU/ml of EPO (Recormon Epoetin, Roche). The cells were plated atapproximately 1.5×10⁵ cells/well in 96-well plates (Costar), andcultured in a humidified 37° C. incubator containing 5% CO₂-95% air.

In the next day, different concentrations of compound A (0, 0.1, 0.5,2.5, 12.5 and 20 μg/ml) were added to the cells, respectively, andincubated for 4 days. The relative levels of hemoglobin were determinedusing DAF-based hemoglobin colorimetry assay according to Kaiho (1985)and Worthington (1985) with minor modifications. In brief, cells werewashed with PBS and lysed in Nonidet™ P 40 (NP-40, Sigma-Aldrich) (0.01%(v/v), 50 μl/well). Cells were then incubated with4,5-diaininofluorescein (DAF, Sigma-Aldrich) (100 μg/ml, 100 μl/well),and 30% hydrogen peroxide (Sigma-Aldrich) (6 μl/well) for 5-10 min. Theabsorbance at 610 nm was measured by Victor 2 1420 Multilable Counter(Wallac, PerkinElmer). Results were expressed as relative index±S.E.(n=6). Statistical significance was evaluated by Student's t test(**P<0.01 versus control group (0 μg/ml)). It was found that compound Aat concentrations between about 0.1 to about 20 μg/ml significantlyenhanced hemoglobin formation (FIG. 1).

Example 3 Compound A Activates Erythroid Progenitor Cells in CellCulture

To investigate the effect of “Compound A” on erythroid progenitor cells,burst-forming-units-erythroid (BFU-E) assay was conducted according tothe procedures described in Corazza et al. (2004) and Jin el al. (2003)with modifications. Briefly, 6-week-old C57BL/6JNar1 mice were purchasedfrom the National Laboratory Animal Center (NLAC, Taiwan). Bone marrowcells were isolated from the mice, and the cell suspension was adjustedto about 8.3×10⁴ cells/ml in a-MEM containing 15% (v/v) FBS (Gibco), 1%(v/v) BSA (Sigma-Aldrich), 0.8% (w/v) methylcellulose (Sigma-Aldrich),10 μM 2-mercaptoethanol (Sigma-Aldrich), 2 U/ml Epo (Recormon Epoetin,Roche), and 10 ng/ml IL-3 (Sigma-Aldrich). Cells were plated atapproximately 7.5×10⁴ cells/well in 24-well plates (Falcon). They wereincubated with different concentrations of Compound A (0, 0.1, 0.5, 2.5and 12.5 μg/ml), respectively, for 9 days in a humidified 37° C.incubator containing 5% CO₂-95% air. The colonies were stained with3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (50 μg/50μl/well) (MTT; Sigma-Aldrich). Plates were photographed while coloniesof more than 50 cells were counted. Results were expressed as mean±S.E.(n=3). Statistical significance was evaluated by Student's /test(*P<0.05 and **P<0.01 versus control group (i.e., 0 concentration)).Compound A at concentrations of 0.5˜12.5 μg/ml significantly increasedthe number of BFU-E colonies of bone marrow cells (FIG. 2). The resultsindicated that Compound A enhanced erythropoiesis by increasinghemoglobin formation and activating erythroid progenitor cells.

Example 4 Compound A Enhances Kidney Expression of Epo

C57BL/6JNar1 mice, 8-10 weeks of age, were purchased from the NationalLaboratory Animal Center (NLAC, Taiwan). The kidney slices of mice wereprepared as previously described with modifications (Parrish el al.,1995, Obatomi et al., 1998 and De Kanter et al., 1999). Briefly, micewere sacrificed by cervical dislocation and the kidneys were excised andimmediately kept in gassed (95% O₂ and 5% CO₂) ice-cold sterileKrebs-HEPES buffer (pH 7.4) containing 20 mM HEPES (USB), 128 mM sodiumchloride (Sigma-Aldrich), 2.5 mM potassium chloride (Sigma-Aldrich), 2.7mM calcium chloride (Sigma-Aldrich), 1 mM magnesium chloride(Sigma-Aldrich) and 16 mM D-glucose (Sigma-Aldrich). The kidneys weredecapsulated and cored perpendicular to the cortico-papillary axis.Kidney slices (250 μm) were prepared in microslicer (D.S.K microslicer,DRK 1000, Dosaka EM Co) filled with gassed (95% O₂: 5% CO₂) ice-coldsterile Krebs-HEPES buffer. Kidney slices were collected and stored onice in gassed (95% O₂, 5% CO₂) ice-cold sterile Krebs-HEPES buffer andused within 10 min of slice preparations.

Kidney slices were preincubated in a 24-well plate (Falcon) containing1.0 ml/well of gassed (95% O₂, 5% CO₂) fresh media (DMEM/F12(Gibco/Invitrogen) containing 15 mM HEPES (USB), 20 mM sodiumbicarbonate (Sigma-Aldrich)) and 5% (v/v) FBS (Gibco) for 90˜120 min at37° C. in a 95% O₂: 5% CO₂ atmosphere chamber. Afterwards, kidney sliceswere incubated with different concentrations of Compound A (0, 0.6, 2.5,10, 40 and 100 μg/ml), respectively, for 16 hr at 37° C. in a 50% O₂: 5%CO₂: 45% N₂ atmosphere chamber. Ribonucleic acids (RNA) of kidney sliceswere extracted by RNA-Beel RNA isolation solvent (Tel-test). Total RNA(5 μg) was used to prepare complementary deoxyribonucleic acid (cDNA)using MMLV reverse transcriptase (Promega). The reverse-transcribed cDNAsamples were analyzed by polymerase chain reaction (PCR) using ABIGeneAmp™ system 2700 (Applied Biosystems) with primers targeting towardmouse Mactin (SEQ ID NOs: 1 and 2) and erythropoietin (Epo) (SEQ ID NOs:3 and 4) (Table 1). Results were compared with control group (0 μg/ml)and expressed as relative index. The results indicated that Compound Aincreased the expression of Epo in kidney tissue (FIG. 3).

TABLE 1 Primer Sequence ID NO. β-actin-F GTGGGCCGCCCTAGGCACCA SEQ IDNO:1 β-actin-R TGGCCTTAGGGTTCAGGGGG SEQ ID NO:2 Epo-FAATGGAGGTGGAAGAACAGG SEQ ID NO:3 Epo-R ACCCGAAGCAGTGAAGTGA SEQ ID NO:4GAPDH-F TGGCATCGTGGAAGGGCTCA SEQ ID NO:5 GAPDH-R GGAAGAATGGGAGTTGCTGTSEQ ID NO:6 HGF-F CTTGGCATCCACGATGTTCAT SEQ ID NO:7 HGF-RTGGTGCTGACTGCATTTCTCA SEQ ID NO:8

Example 5 Compound A Enhances Hepatocyte Expression of Epo

C57BL/6JNar1 mice, 8-10 weeks of age, were purchased from the NationalLaboratory Animal Center (NLAC, Taiwan). The hepatocytes were isolatedas previously described with modifications (Kreamer el al., 1986).Briefly, mice were anaesthetized with Avertin (2% (w/v)2,2,2-tribromoethanol, i.p., 300 mg/Kg body weight). and the liver wasperfused in situ through portal vein with a calcium-free Hank's balancedsalt solution (HBSS) containing 0.5 mM Ethyleneglycol-bis(2-aminoethylether)-N,N,N,N′-tetraacetic acid (EGTA;Sigma-Aldrich). The flow rate was 1 ml/min with the perfusate exitingvia the severed inferior vena cava. After 10 min of perfusion, HBSScontaining 0.05% (w/v) collagenase (Type II; Worthington BiochemicalCorporation) and 1 mM calcium chloride (Sigma-Aldrich) was introducedinto the perfusion apparatus. The perfusion was continued for another 10min at the same flow rate. The partially digested liver was removed,placed in a 60-mm culture dish (BD Falcon) containing HBSS, and gentlyminced to open the liver capsula. The minced liver suspension wasfiltered through a nylon mesh (about 300 μm pore size), centrifugated at50×g for 3 min to obtain a cell pellet. The percoll (GE Healthcare)medium of a density of 1.06 g/ml was prepared. The cell pellet wassuspended in DMEM medium (Gibco/Invitrogen), centrifuged with thepercoll medium at 50×g for 10 min at 4° C., washed once with DMEM mediumand followed by centrifugation at 50×g for 2 min to obtain hepatocytes.

The isolated hepatocytes were plated in 6-well plates (Costar) atapproximately 6×10⁵ cells/well in DMEM medium (Gibco/Invitrogen)containing 10% FBS (Gibco) in a humidified 37° C. incubator containing5% CO₂-95% air. After 3 hr, the medium was removed, and the cells wereincubated with different concentrations of compound A (0, 0.1, 0.5, 2.5and 12.5 μg/ml), respectively, for 24 hours. The RNA of hepatocyte wasextracted by RNA-Bee™ RNA isolation solvent (Tel-test) for reversetranscription, and the transcripts analyzed by PCR as previouslydescribed. The results indicated that Compound A enhanced the expressionof Epo in hepatocytes (FIG. 4).

Example 6 Compound A Enhances Kidney Function in a Cisplatin-InducedAcute Renal Failure Animal Model

The effects of Compound A treatment on cisplatin- induced acute renalfailure animal model were investigated. C57BL/6JNar1 mice (8-week-old)were purchased from the National Laboratory Animal Center (NLAC,Taiwan). Cisplatin (Sigma-Aldrich) was dissolved in normal saline (0.9%(w/v) sodium chloride, Sigma-Aldrich) at a concentration of 1 mg/ml, andinjected into mice intraperitoneally (i.p.) according to the followingschedule: day 1, 7 mg/kg; day 5, 6 mg/kg; and day 9, 6 mg/kg. Forcisplatin-untreated normal mice group, normal saline injections ofcorresponding volumes were given i.p. in parallel. At day 13, serumsamples were collected and blood urea nitrogen (BUN) contents wereassayed. Cisplatin-injected mice with BUN levels more than 100 mg/dl andthe cisplatin-untreated normal mice were further divided into 5 groups,6 mice per group, for feeding with Compound A for 2 weeks: (1) Normalgroup: Mice were injected with normal saline and followed by feedingwith a standard diet; (2) Control group: Mice were injected withcisplatin and followed by feeding with a standard diet; (3) Pomu 10mg/kg/day group: Mice were injected with cisplatin and followed byfeeding with Compound A at the dose of 10 mg/kg/day; (4) Pomu 30mg/kg/day group: Mice were injected with cisplatin and followed byCompound A at the dose of 30 mg/kg/day; (5) Pomu 90 mg/kg/day group:Mice were injected with cisplatin and followed by Compound A treatmentat the dose of 90 mg/kg/day.

Blood samples were collected from the retro-orbital sinus of mice at day0, 5, 10, 13, 18, 23 and 28. Red blood cell (RBC) concentrations weredetermined by complete blood cell count using Sysmex Kx-2 I hematologyanalyzer. Blood urea nitrogen (BUN) levels were determined by ureaseGLDH method (Urea FS, DiaSys). Results were expressed as mean±S.E.(n=5˜6). Statistical significance was evaluated by Student's/test(##P<0.01 versus normal group and **P<0.01 versus control group). Theresults indicated that Compound A treatment at concentrations of 30˜90mg/kg/day increased the number of red blood cells (RBCs) (FIG. 5) anddecreased serum BUN levels (FIG. 6 ). At day 23, Compound A atconcentrations between 30˜90 mg/kg/day significantly increased thenumber of RBCs (FIG. 7) while decreasing serum BUN levels (FIG. 8). Thehorizontal arrow in FIGS. 7 and 8 represents Compound A treatment(mg/Kg/day). The data indicated that the Compound A treatment enhancedrenal functions and reduced anemia in a cisplatin-induced acute renalfailure animal model. The results were in agreement with the ability ofthe Compound A in enhancing erythropoiesis.

Example 7 Compound A Increases Liver Expressions of Epo and HepatocyteGrowth Factor in a Cisplatin-induced Acute Renal Failure Animal Model

At the end of the experiments described in EXAMPLE 6 (i.e., day 28),mice were sacrificed, kidney and liver tissues were removed, and bonemarrow cells were isolated. RNA from tissues and bone marrow cells wereextracted by RNA-Bee™ RNA isolation solvent (Tel-test). Total RNA (5 μg)was used to prepare cDNA using MMLV reverse transcriptase (Promega). Thereverse-transcribed cDNA samples were analyzed by PCR using ABI GeneAmp™system 2700 (Applied Biosystems) and the SYBR Green Master Mix kit(Applied Biosystems) using primers targeting toward mouse glyceraldehyde3-phosphate dehydrogenase (GAPDH) (SEQ ID NOs: 5 and 6), erythropoietin(Epo) (SEQ ID NOs: 3 and 4) and hepatocyte growth factor (HGF) (SEQ IDNOs: 7 and 8) (Table 1). Results were expressed as relative index ±S.E.(n=5˜6). Statistical significance was evaluated by Student's t test (##P<0.01 versus normal group and *P<0.05, **P<0.01 versus control group).

The results indicated that Compound A at concentrations of 30˜90mg/kg/day significantly increased the expression of Epo in liver in acisplatin-induced acute renal failure animal model as compared to theuntreated control group (FIG. 9). Livers and kidneys are two majororgans that make Epo. Compound A was found to enhance the expression ofEpo in kidney tissue (FIG. 3), hepatocytes (FIG. 4) and liver tissue(FIG. 9). Moreover, compound A (10˜90 mg/Kg/day) treatment also enhancedthe expression of HGF in liver tissue (FIG. 10). The horizontal arrow inFIGS. 9 and 10 represents Compound A treatment (mg/Kg/day).

Example 8 Compound A Activates Erythroid Progenitor Cells in aCisplatin-Induced Acute Renal Failure Animal Model

The isolated bone marrow cells described in EXAMPLE 7 were cultured for12 days in a humidified 37° C. incubator containing 5% CO₂˜95% air forburst-forming-units-erythroid (BFU-E) assay as described above. Resultswere expressed as relative index±S.E. (n=5˜6). Statistical significancewas evaluated by Student's t test (## P<0.01 versus normal group and*P<0.05, **P<0.01 versus control group). It was found that Compound A atconcentrations of 10˜90 mg/kg/day significantly increased the number ofBFU-E colonies of bone marrow cells (FIG. 11). The horizontal arrow inFIG. 11 represents Compound A treatment (mg/Kg/day). The resultsindicated that Compound A activated erythroid progenitor cells andenhanced erythropoiesis.

The compound 2,3,5,4′-tetrahydroxystilbene 2-O-β-D-glucopyranoside,i.e., Compound A, has been found in several plants. According to theliteratures, Compound A has the following pharmacological effects:stimulating melanogenesis by activating tyrosinase (Guan et al., 2008),inhibiting lipid peroxidation (Kimura et al., 1983), having a protectiveeffect on colitis through alleviating oxygen and nitrogen free radicalslevel and down-regulating inducible nitric oxide synthase expression(Wang et al., 2008), having anti-atherosclerosis effect via itsantagonistic effects on oxidation of lipoprotein, proliferation anddecrease in nitric oxide content of coronary arterial smooth cells (Liuet al., 2007), having anti-inflammatory function that is related to theinhibition of cyclooxygenase-2 enzyme activity and expression (Zhang elal., 2007), and are beneficial for Alzheimer disease and cognitiveimpairment (Wang et al., 2007).

U.S. Publication No. 20050042314 (entitled “Extracts of Polygonummultiflorum Thunb., and preparation process and uses of the same)discloses that extract products from the root of Polygomun multiflorumThunb. are biologically active in increasing proliferation, growthand/or differentiation of hematocytes and bone marrow cells. TheFleeceflower root contains numerous chemical components, such aseniodin, chrysophanol, physcion, rhein, chrysophanol anthrone,resveratrol, piceid,2,3,5,4′-tetrahydrostilbene-2-O-β-D-glucopyranoside,2,3,5,4′-tetrahydroxystilbene-2-O-β-D-gluco-pyranoside-2′-O-mo-nogalloylester,2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucopyranosid-e-3″-O-monogalloylester, gallic acid, catechin, epicatechin, 3-O-galloyl(−)-catechin, and3-O-galloyl(−)epicatechin, 3-O-galloyl-procyanidinB-2,3,3′-di-O-galloyl-procyanidin B-2, and .beta.-sitosterol. The U.S.Publication No. 20050042314 is silent as to which chemical component(s)is active.

As described above, Compound A has been proved to be biologically activein increasing Epo production and enhancing erythropoiesis and kidneyfunctions. The glucosyl group in Compound A may be replaced bysaccharides such as dihydroxyacetone, galactose, glyceraldehydes,threose, xylose, mannose, ribose, ribulose, xylulose, tagatose, psicose(allulose), fructose, sorbose, rhamnose, erythrose, erythrulose,arabinose, lyxose, allose, altrose, gulose, idose, talose, and anycombinations thereof.

It is well known that Epo stimulates erythropoiesis, and that Epo isrequired for erythropoiesis (Moritz el al., 1997). It has also beenreported that Epo has renoprotective effects (Sepodes el al., 2006 andArcasoy, 2008). In addition to increasing Epo production, Compound Aenhances erythropoiesis and kidney functions.

HGF is a multifunctional cytokine that is related to cell survival,growth, motility and morphogenesis, tissue regeneration, protection andrepair. It has been reported that HGF activates signal transduction fromEpo receptor (Iguchi et al., 1999 and Isobe et al., 2006). HGF has beenshown to have therapeutic potential for allergic inflammation (Ito etal., 2008), osteoarthritis, rheumatoid arthritis, muscular dystrophy,muscular atrophy, skin ulcer, burn, scleroderma, crush syndrome(Funakoshi and Nakamura, 2003), cerebrovascular diseases such astransient ischemic attack and stroke, neurodegenerative diseases such asamyotrophic lateral sclerosis, Alzheimer's disease and Parkinson'sdisease, spinal cord injury, diabetic retinopathy, peripheralneuropathy, spinal canal stenosis, deafness (Ftnakoshi and Nakamura,2003 and Takeo et al., 2007), acute hepatitis, liver cirrhosis,fulminant hepatitis, fatty liver, surgical treatments for livertransplantation, partial resection and ischemia (Funakoshi and Nakamura,2003 and Mizuno and Nakamura, 2007), kidney fibrosis, acute renalfailure, chronic renal failure such as nephritic syndrome andobstructive nephropathy, surgical treatments for renal transplantationand ischemia, diabetic nephropathy (Matsumoto and Nakamura, 2001;Funakoshi and Nakamura, 2003 and Liu, 2004), acute pneumonia, pulmonaryfibrosis, surgical treatments for lung transplantation, partialresection and ischemia (Funakoshi and Nakamura, 2003),inflammation-mediated heart diseases such as cardiac allograft rejectionand myocarditis, cardiovascular diseases such as angina, cardiacinfarction, cardiomyopathy and atherosclerosis obliterans (Funakoshi andNakamura, 2003 and Isobe et al., 2006), intestinal mucosal injury,inflammatory bowel disease (Ido et al., 2005), gastric ulcer anddiabetes mellitus (Funakoshi and Nakamura, 2003).

Compound A has a structure based on 3,4′,5-trihydroxy-trans-stilbene,which is a natural compound possessing numerous biological activities,including renoprotection via nitric oxide dependent and/or antioxidantmechanisms (Sharma et al., 2006; Shankar et al., 2007; Do Amaral et al.,2008 and Orallo, 2008). Compound A was found to be superior to3,4′,5-trihydroxy-trans-stilbene in enhancing renal functions andimproving anemia (data not shown).

rHuEpo and NESP have been used in intravenous (i.v.) or subcutaneous(s.c.) injection for treating anemia of CKD, anemia in cancer patientsreceiving chemotherapy, anemia in zidovudine-treated patients infectedwith human immunodeficiency virus (Fisher, 2003). It has beenrecommended that the administration be given more than once per week formaintenance therapy. The cost, the inconvenience due to frequentinjections (parenteral administration) and development of anti-Epoantibodies due to inherent antigenicity of rHuEpo all point to a needfor developing an improved therapeutic agent. Oral administration ofCompound A, one of 3,4′,5-trihydroxy-trans-stilbene analogs, canincrease Epo formation, enhances erythropoiesis and kidney functions,and reduces anemia.

The compound 3,4′,5-trihydroxy-trans-stilbene has been reported to haverenoprotective effects via nitric oxide dependent and/or antioxidantmechanisms. Compound A is superior to 3,4′,5-trihydroxy-trans-stilbenein enhancing erythropoiesis and kidney functions. Moreover, Compound Ahas the advantages that recombinant Epo does not have: An oraladministration of Compound A does not pose inherent antigenicity, whichrecombinant Epo has. In addition, the route of administration of rEPO isinconvenient because it requires frequent injections. Compound A may bebeneficial for disorder-associated anemia, such as CKD, cancer, acuteand chronic infections, autoimmune, inflammation, chronic rejectionafter solid-organ transplantation, chemotherapy-induced anemia, andtissue ischemia.

The foregoing description of the exemplary embodiments of the inventionhas been presented only for the purposes of illustration and descriptionand is not intended to be exhaustive or to limit the invention to theprecise forms disclosed. Many modifications and variations are possiblein light of the above teaching.

The embodiments and examples were chosen and described in order toexplain the principles of the invention and their practical applicationso as to enable others skilled in the art to utilize the invention andvarious embodiments and with various modifications as are suited to theparticular use contemplated. Alternative embodiments will becomeapparent to those skilled in the art to which the present inventionpertains without departing from its spirit and scope. Accordingly, thescope of the present invention is defined by the appended claims ratherthan the foregoing description and the exemplary embodiments describedtherein.

Some references, which may include patents, patent applications andvarious publications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entiretiesand to the same extent as if each reference was individuallyincorporated by reference.

LIST OF REFERENCES

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1. A method for enhancing erythropoietin formation in a subject in needthereof comprising the step of administering to the subject an effectiveamount of a compound of the formula:

wherein R is a glycosyl group.
 2. The method of claim 1, wherein theglycosyl group is at least one selected from the group consisting ofdihydroxyacetone, glucose, galactose, glyceraldehydes, threose, xylose,mannose, ribose, ribulose, xylulose, tagatose, psicose (allulose),fructose, sorbose, rhamnose, erythrose, erythrulose, arabinose, lyxose,allose, altrose, gulose, idose and talose, and any combinations thereof.3. The method of claim 1, wherein the compound of the formula (I) isadministering to a tissue selected from the group consisting of bonemarrow, a liver tissue and a kidney tissue, and any combinationsthereof.
 4. A method for treating a disease and/or disorder in a subjectthat would benefit from erythropoietin treatment comprising the step ofadministering to the subject an effective amount of a compound of theformula:

wherein R is a glycosyl group.
 5. The method of claim 4, wherein thedisease is at least one selected from anemia, a renal failure, anderythropoiesis deficiency-related diseases.
 6. The method of claim 4,wherein the disorder would benefit from an erythropoietin-relatedprotective effect.
 7. The method of claim 6, wherein theerythropoietin-related protective effect is at least one selected fromneuroprotection, cardiovascular protection, renoprotective effect, andprotections from tissue ischemia and ischemia/reperfusion injury.
 8. Amethod for enhancing erythropoiesis in a subject in need thereofcomprising the step of administering to the subject an effective amountof a compound of the formula:

wherein R is a glycosyl group.
 9. The method of claim 8, wherein theglycosyl group is at least one selected from the group consisting ofdihydroxyacetone, glucose, galactose, glyceraldehydes, threose, xylose,mannose, ribose, ribulose, xylulose, tagatose, psicose (allulose),fructose, sorbose, rhamnose, erythrose, erythrulose, arabinose, lyxose,allose, altrose, gulose, idose and talose, and any combinations thereof.10. A method for enhancing kidney function in a subject in need thereofcomprising the step of administering to the subject an effective amountof a compound of the formula:

wherein R is a glycosyl group.
 11. The method of claim 10, wherein theglycosyl group is at least one selected from the group consisting ofdihydroxyacetone, glucose, galactose, glyceraldehydes, threose, xylose,mannose, ribose, ribulose, xylulose, tagatose, psicose (allulose),fructose, sorbose, rhamnose, erythrose, erythrulose, arabinose, lyxose,allose, altrose, gulose, idose and talose, and any combinations thereof.12. A method for enhancing the expression of hepatocyte growth factor ina subject in need thereof comprising the step of administering to thesubject an effective amount of a compound of the formula:

wherein R is a glycosyl group.
 13. The method of claim 12, wherein theglycosyl group is at least one selected from the group consisting ofdihydroxyacetone, glucose, galactose, glyceraldehydes, threose, xylose,mannose, ribose, ribulose, xylulose, tagatose, psicose (allulose),fructose, sorbose, rhamnose, erythrose, erythrulose, arabinose, lyxose,allose, altrose, gulose, idose and talose, and any combinations thereof.14. A method for treating a disease and/or disorder in a subject thatwould benefit from hepatocyte growth factor treatment comprising thestep of administering to the subject an effective amount of the compoundof the formula:

wherein R is a glycosyl group.
 15. The method of claim 14, wherein theglycosyl group is at least one selected from the group consisting ofdihydroxyacetone, glucose, galactose, glyceraldehydes, threose, xylose,mannose, ribose, ribulose, xylilose, tagatose, psicose (allulose),fructose, sorbose, rhamnose, erythrose, erythrulose, arabinose, lyxose,allose, altrose, gulose, idose and talose, and any combinations thereof.16. The method of claim 14, wherein the disease and/or disorder is atleast one selected from the group consisting of a liver disease, akidney disease, a lung disease, a cardiovascular disease, a digestivedisease, a neuronal disease, a bone disease, a joint disease, a musculardisease, a skin disease, crush syndrome, allergic inflammation, and anycombinations thereof.